Novel antimicrobial polypeptides and methods of use

ABSTRACT

Antimicrobial compounds and compositions and uses thereof, including the treatment and prevention of bacterial infections are described. The compounds and compositions include lantibiotic polypeptides and the nucleic acid sequences encoding the polypeptides. The compounds and compositions are useful as antimicrobials in antibiotic pharmaceutical preparation and as an antimicrobial or antiseptic dentifrice.

BACKGROUND OF THE INVENTION

[0001] The subject invention was made with government support under aresearch project supported by National Institute of Dental ResearchGrant No. DE04529. The government has certain rights in this invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] Not applicable.

[0003] The subject invention concerns novel polypeptides and nucleicacid sequences encoding those polypeptides. The polypeptides are relatedto bacteriocins, e.g. mutacins, produced by microbes for providing aselective advantage for the microbe. The invention includes methods ofuse which exploit the advantageous activities or properties of thepolypeptides or nucleic acid sequences.

[0004] The phenotypically similar bacteria collectively known as themutans streptococci are considered major etiologic agents responsiblefor dental caries. The species most commonly associated with humandisease is Streptococcus mutans. Pathogenicity of S. mutans includes theability to produce antimicrobial substances generally referred to asbacteriocin-like inhibitory substances (BLIS) or bacteriocins.Bacteriocins produced by Streptococcus mutans are known as mutacins.These substances are produced by microorganisms to provide a selectiveforce necessary for sustained colonization in a milieu of densely packedcompeting organisms found in dental plaque.

[0005] To date, most bacteriocins remain only partially characterizedbecause they are made in small quantities and only under specialcultivation conditions. In addition, mutacins are known to be difficultto isolate from liquid medium. The spectrum of activity and chemical andphysical properties of mutacins can vary widely.

[0006] Certain bacteriocin peptides or mutacins produced by S. mutansgroup II have recently been characterized as belonging to a group ofpeptides called lantibiotics. Novak, et al. (1996) Anal. Biochem.236:358-360. Lantibiotics are polycyclic peptides which typically haveseveral thioether bridges, and which can include the amino acidslanthionine or β-methyllanthionine. In addition, lantibiotics cancontain a,β-unsaturated amino acids such as 2,3-didehydroalanine and2,3-didehydro-2-aminobutyric acid, which are the products ofpost-translational modification of serine and threonine residues,respectively.

[0007] Certain lantibiotics have demonstrated antibiotic activity,mainly against Gram-positive bacteria (Bierbaum and Sahl (1993) Int. J.Med. Microbiol. Virol. Parasitol. Infect. Dis. 278:1-22). Nisin andepidermin are the best known examples of the 20 or so lantibiotics whichhave been identified to date. They are ribosomally synthesized asprepropeptides that undergo several post-translational modificationevents, including dehydration of specific hydroxyl amino acids andformation of thioether amino acids via addition of neighboring cysteinesto didehydro amino acids. Further post-translational processing involvescleavage of a leader sequence, which can be coincident with transport ofthe mature molecule to the extracellular space. A mature lantibioticmolecule is usually about 20 to 35 residues in which the thioetherlinkages result in cyclical segments that provide a substantial degreeof rigidity to the rodlike structure.

[0008] Current evidence indicates that the biological activity ofcertain lantibiotics, e.g., those known as “type A” lantibiotics,depends on the association of a number of molecules with the membrane ofa target bacterium to form ion channels, thereby resulting indesynergization. Rapid loss of all biosynthetic processes occurs,resulting in death of the target cell. Other lantibiotics known as “typeB” lantibiotics, can exert their effect by specifically inhibitingcertain enzymes.

[0009] The genetics of lantibiotic production have been studied inseveral species of bacteria. In general, it has been found that thestructural gene for the preprolantibiotic is clustered with genes whichencode products responsible for post-translational modifications of thelantibiotic. In certain instances, these genes are known to form anoperon or operon-like structure (e.g., Schnell, et al. (1992) Eur. J.Biochem. 204:57-68). Production of lantibiotics also can requireaccessory proteins, including processing proteases, translocators of theATP-binding cassette transporter family, regulatory proteins, anddedicated producer self-protection mechanisms. At least seven genes havebeen shown to be involved in epidermin biosynthesis.

[0010] Lantibiotic properties have been exploited in certain productsthat are commercially available. The antibiotic, nisin, has beendeveloped as a food preservative which has been given “GenerallyRecognized as Safe (GRAS)” status by the federal Food and DrugAdministration (FDA). It is employed in this fashion in more than 40countries in preference to nitrites and nitrates. The oral toxicity ofthis compound, and presumably other lantibiotics, is very low in rats(LD₅₀=7 g/kg; Hurst, (1981) Adv. Appl. Microbiol. 27:85-123). Otherapplications for nisin, including its use as a mouth rinse (Howell, etal. (1993) J. Clin. Periodontal 20:335-339), are actively being examinedby a large number of laboratories.

[0011] The discovery of new lantibiotic compounds having antibioticactivity can be particularly important in view of the increasedresistance to presently available antibiotics that have been shown inrecent years to have developed in certain pathogenic microorganisms.Novel lantibiotic compounds having unique or superior activity againstparticularly virulent pathogenic bacteria are advantageous in providingnew weapons in the arsenal against bacterial infection.

BRIEF SUMMARY OF THE INVENTION

[0012] The present invention is summarized in that a novel lantibiotic,here identified as mutacin 1140, has been identified from Streptococcusmutans. The lantibiotic mutacin 1140 has a wide spectrum of activityagainst bacteria of several genera.

[0013] The present invention is also summarized by the identificationand sequencing of genetic elements associated with the synthesis of thelantibiotic mutacin 1140 in its native host. These genetic elementsfacilitate the synthesis of the lantibiotic mutacin 1140 in othermicrobial hosts.

[0014] It is yet another object of this invention to provide a method oftreatment for human beings or other animals having bacterial infectionor infestation. A particular object of the invention is to providetreatment of an animal against infection or colonization by a pathogenicorganism that can cause dental caries or other oral pathogenic events.The method of treatment comprises contacting a target microbe with aneffective amount of the compound, or a composition comprising thatcompound, to kill, inhibit, or otherwise control the growth orproliferation of the target microorganism.

[0015] Still further, the nucleic acid sequences of the subjectinvention can be employed in standard genetic engineering procedures totransform appropriate host cells for producing polypeptides according tothe subject invention. Other uses for the subject polypeptide orpolynucleotide sequences will be recognized by ordinarily skilledartisans in view of currently available knowledge and the descriptionprovided herein.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0016] FIGURE 1 shows a proposed secondary structure of a polypeptideaccording to the subject invention. Abbreviations of amino acids includethe following: ala-S-ala=lanthionine; abu-S-ala=3-methyl anthionine;dha=α,β-didehydroalanine; dhb=α,β-didehydrobutyrine. The C-terminalcysteine is added to dha in position 19 and oxidized to yield aS-aminovinyl-D-cysteine.

DETAILED DESCRIPTION OF THE INVENTION

[0017] Described herein is a novel antibiotic first identified from astrain of Streptococcus mutans designated JH1140. The antibiotic, heretermed mutacin 1140, like other lantibiotics, is a polycyclic peptidewhich is the product of post translational modification of a precursorprotein translated from a single gene transcript in the host organism.The identified molecular structure of mutacin 1140 is illustrated inFIGURE 1.

[0018] Lactate-dehydrogenase deficient mutants of Streptococcus mutanshave been studied for their potential use in replacement therapy fordental caries. Without the trait of LDH, fermentation of carbohydratesby this microorganism employs alternate pathways for pyruvate metabolismthat yields significant amounts of neutral end products, and thus LDHdeficient strains exude less total acids into the environment. As aresult, LDH deficient mutants of this bacteria are less cariogenic.Thus, these bacterias are being studied as an effector strain forreplacement therapy for dental caries. However, in order to be aneffective replacement strain, strains must demonstrate superiorcompetitive colonization properties in order to compete against otherstrains of the species and to prevent subsequent recolonization bywild-type strains. Accordingly, effort has been conducted to findstrains which have both superior colonization properties as well as anLDH-deficiency phenotype.

[0019] One of the evolutionary strategies utilized by microorganisms forenhanced competitiveness with competing strains is the synthesis ofantibiotic agents to which competitive strains are sensitive. It wasfound here that a strain of S. mutans, previously called JH1000, anethyl methane sulfonate-induced mutant called JH1005, and a spontaneousmutant of that strain, known as JH1140, which have been previouslyreported to have good colonization properties, produced a potent broadspectrum bacteriocin-like inhibitory substance, referred to as a BLIS.As described below, the BLIS was found to inhibit the growth ofrepresentative strains of a wide variety of bacterial species. Inaddition, virtually all known Streptococcus mutans strains tested weresensitive to the BLIS substance.

[0020] Analysis of isogenic mutants of these strains demonstrated goodcorrelation between BLIS production and colonization potential in both arodent model and human subjects. Utilizing genetic methods, thetranscript responsible for the BLIS activity has been identified andsequenced. Presented as Sequence ID NO: 1 below is the genomic copy ofthe single transcript encoding the peptide responsible for the BLISactivity, the gene being named here lanA. Identified as Sequence ID NO:2 below is the deduced amino acid sequence of the transcript produced byan open reading frame present in Sequence ID NO: 1. Sequence ID NO: 2 isthe pre-protein form which, after proteolytic cleavage and otherprocessing by other factors present in the host organism, results in thesynthesis of mutacin 1140 as shown in FIGURE 1.

[0021] The proper synthesis of mutacin 1140 in the host microorganismrequires the presence of other enzymes to properly process the precursorform of the protein into the effective and active form of the peptideantibiotic. The gene encoding one of those enzymes, here designatedlanB, has also been cloned and sequenced and is presented as Sequence IDNO: 3 below. Sequence ID NO: 4 below presents the deduced amino acidsequence of the open reading frame contained in Sequence ID NO: 3.

[0022] As used herein, the term “mutacin 1140” is intended to apply tothe peptide antibiotic produced by Streptococcus mutans strain 1140, aswell as related peptides produced by minor insertions, deletions orother variants which do not detract from the biological efficacy of thelantibiotic. It should be understood that while the chemical structurepresented in FIGURE 1 is believed correct, that due to limitation in theanalytical techniques used to date to elucidate the structure of themolecule, it is possible that there may be some minor differencesbetween the structure of FIGURE 1 and the actual structure of themolecule produced by the bacteria, particularly at the carboxyl-end ofthe peptide. It is intended that the term mutacin 1140 describes theactual molecule in the event there are such minor differences. It isalso anticipated that other evolutionarily-related strains ofStreptococcus mutans, or closely related strains of other species, couldproduce allelic variations of this same lantibiotic and the term mutacin1140 is intended to cover those as well.

[0023] It has been found that mutacin 1140 is an antibiotic with anevolutionary relationship to another antibiotic known as epiderminproduced by Staphylococcus epidermidis. The genetic sequence presentedbelow, derived from a mutant strain JH1005 derived from JH1000, includessequences with a high degree of homology to epiA, B and D, which aregenes previously sequenced from Staphylococcus epidermidis and found tobe involved in the biosynthesis of the antibiotic epidermin. The lanAand lanB genes presented herein are believed to be roughly analogous tothe epiA and epiB genes associated with the antibiotic epidermin.

[0024] The antibiotic polypeptide mutacin 1140 of the present inventioncan be isolated from the culture medium in which its native hostorganism, i.e., a Streptococcal organism, has been grown in culture,followed by isolation of the polypeptide antibiotic from the culturemedium. In addition, the presentation of the lanA and lanB codingsequences below allows for the construction of artificial genes encodingthese sequences which can be transformed into other Streptococcalspecies or strains of other bacterial species. Two Streptococcal strainswhich produce the mutacin 1140 antibiotic have been deposited with theAmerican Type Culture Collection, Rockville, Md., as Accession Numbers55676 (JH1140) and 55677 (JH1000). The mutacin 1140 antibiotic can berecovered from these strains, or other related strains of Streptococcalspecies into which the genetic capability to synthesize mutacin 1140 isintroduced using the information from Sequence ID NOS: 1 through 4below.

[0025] A potential complexity in the introduction of the phenotype ofproduction of mutacin 1140 into a new strain is the fact that thepeptide undergoes post-translational modifications by other geneticelements in the host strain. As mentioned above, the lanB gene presentedbelow is a necessary, but not sufficient, genetic component for the posttranslational modification. The other post translational modificationgenes are contained within the genome of strain JH1140 as depositedabove. By performing a random-type genetic transfer experiment of DNAfrom mutacin 1140-competant hosts into other Streptococcal strains, onecan readily identify what other genetic components are necessary, inaddition to lanA and lanB presented below, to achieve the fully matureand biologically active form of mutacin 1140 produced by the nativeproducing Streptococcal host strains. Such procedures are within theordinary level of skill in the art. Once identified, these other geneticcomponents can be transferred together with lanA and lanB into a newhost which would then produce mutacin 1140.

[0026] It is also specifically envisioned that mutacin 1140 can besynthesized ex vivo. A number of techniques exist for the synthesis ofpeptide molecules by a relatively conventional organic chemicaltechniques. For example, solid phase polypeptide synthesis permits thecreation of peptides, and that technology has evolved to the point wherepeptides of the size of mutacin 1140 can readily be synthesized outsideof a microbial host.

[0027] It is envisioned that the mutacin 1140 antibiotic will be usefulgenerally as an antibiotic. Since the antibiotic is produced by a commonStreptococcal strain present in human mouths, it is expected to berelatively non-toxic to human species. This conclusion is furtherbuttressed by its analogous characteristic to existing antibiotics, suchas epidermin, which are known to be quite non-toxic to mammals. In itsmethod of use, the mutacin 1140 is applied to the area in which it isdesired to inhibit microbial growth. A carrier may be used to assistdelivery of the antibiotic. In such delivery, it is desired to deliveran effective amount of the lantibiotic, such an effective amount beingreadily determinable by empirical testing to determine what amount oflantibiotic achieves the desired level of microbial inhibition.

EXAMPLE 1 Purification of a Lantibiotic

[0028] A lantibiotic was purified from Streptococcus mutans JH1140 usingthe following procedure:

[0029] Four liter batches of Todd-Hewitt broth (THB; Difco) containing0.5% LE agarose (SeaKem) were sterilized and poured into 90 mm petriplates. The plates were dried overnight at 37° C. A pure culture ofJH1140 on a brain-heart infusion starter plate was used to inoculate 3ml of THB and the cell suspension was vortexed for 10 sec. About 0.3 mlof the cell suspension was spread on the surface of a BHI agar plate andincubated overnight at 37° C. in a candle jar.

[0030] A 10-pronged inoculator was ethanol-flame sterilized and used toinoculate JH1140 from the spread plate prepared as above into evenlyspaced stabs in the plates prepared as above. The plates were incubatedin candle jars at 37° C. for 72 hours. The agar was scraped from theplates entirely and placed into centrifuge bottles. The bottles werestored overnight at −20° C.

[0031] The bottles were then centrifuged at room temperature for 60 min.at 4,000 rpm in a Sorvall RC2B centrifuge and then for an additional 30min. at 8,000 rpm. The supernatant was recovered and passed throughWhatman #1 filter paper in a Buchner funnel.

[0032] To the filtered extract (ca. 3,000 ml) in a 4 L beaker, 100 ml ofchloroform was added. The solution was placed on a magnetic stirrer andagitated at high speed for 120 min. The stir bar was removed and thesolution was allowed to stand overnight undisturbed.

[0033] The aqueous (upper) phase was aspirated off and discarded. Thechloroform layer, containing a milky white flocculent, was divided into50 ml conical centrifuge tubes and centrifuged at ca. 4,000 rpm for 8min. Residual aqueous material was removed by aspiration. The clearchloroform layer was removed using a Pasteur pipette, leaving theflocculent which was washed 2 times with 5 ml of chloroform. Chloroformwas evaporated from the flocculent using a stream of nitrogen gas; thetube was placed in a 45-50° C. water bath during this process to promoteevaporation.

[0034] The dried residue was dissolved in 0.5 ml of 50% ethanol;undissolved material was removed by centrifugation at 13,000×g for 2min. at room temperature. The clarified fraction including thelantibiotic was then stored at −20° C. until further use.

EXAMPLE 2 Bioassay of Lantibiotic Activity

[0035] Antimicrobial activity of the lantibiotic was determined by thefollowing procedure:

[0036] 5 ml of THB were inoculated with S. rattus strain BHT-2(resistant to 1 mg/ml streptomycin); and grown overnight standing at 37°C. 0.02 ml of fractions to be tested for lantibiotic activity wereserially 2-fold diluted in distilled water in microtiter wells. Top agarwas prepared containing BHI broth, 0.75% agar, 1 mg/ml streptomycin, and1:10,000 diluted overnight S. rattus BHT-2 culture from above at 42° C.;0.2 ml was pipetted into each microtiter well. After 5 min. at roomtemperature to allow agar to set, the plate was incubated at 37° C.overnight.

[0037] The minimal inhibitory concentration (MIC) was determined as thereciprocal of the highest dilution of the test fraction which inhibitedgrowth of S. rattus BHT-2 by visual inspection.

EXAMPLE 3 Spectrum of Activity of the Lantibiotic

[0038] Single colonies of the strain producing mutacin 1140 were stabinoculated into brain heart infusion medium and incubated overnight incandle jars at 37° C. Three drops of an overnight Todd-Hewitt brothculture of the indicator strain were mixed with 3 ml of molten top agarand poured evenly over the surface of the plate. After an additional 24hours of incubation, clear zones surrounding the test strain weremeasured.

[0039] Representative strains of various bacteria were tested for theirsensitivity to the inhibitory activity of the mutacin 1140 produced bythe JH1140 strain by using the overlay technique. In addition to S.mutans, most Gram positive organisms were found to be sensitive,including Streptococcus mitis, Streptococcus pyogenes, Staphylococcusaureus, and Actinomyces species. The inhibitory factor inhibited 124 of125 S. mutans strains tested. Gram-negative bacteria were invariablyresistant to inhibition by mutacin 1140. The following table summarizesthe spectrum of activity found for the lantibiotic. The partiallypurified mutacin 1140 had the same spectrum of activity displayed byJH1140, as demonstrated by spotting 5 μl samples on lawns of targetstrains prepared as described above. This is also shown in the table.TABLE 1 Mutacin Sensitivity Assay^(a) Test Strains Indicator StrainTarget Strain JH1140 Strain JH1005 Mutans Streptococci FA1 (a) + +/−BHT-2 (b) + + LM7 (e) + + Ingbritt (c) + + MT-3 (c) + + 10449 (c) + +JC2 (c) + + GS5 (c) + + Pk1 (c) + + Streptococcus salivarius SS2 + +O2 + + O4 + + Streptococcus sanguis Fc-1 + + KJ3 + + Challis − +Streptococcus mitis MT + + RE-7 + + 26 + + Streptococcus pyogenesSTA628 + + Streptococcus faecalis RF − Streptococcus aureus DC3 + +Lactobacillus casei Lac-6 − + Lactobacillus salivarius UCL-37 +Antinomyces israelii X523 + 10048 + Antinomyces naeslundii 12104 + +N16 + + 6-60 B + + Antinomyces viscosus W1528 + T6 + M100 + Micrococcusluteus 207-79 − Bacteroides gingivalis 381 − Wolinella recta 371 −Capnocytophaga sputigena 4 −

[0040] The inhibitory factor was produced in detectable amounts onlyduring early stationary phase and could be recovered from Todd-Hewittbroth cultures of JH1140. The inhibitory factor's effect on otherstrains of S. mutans was bacteriocidal, since loopfuls of agar takenfrom clear zones were found to be sterile. The inhibitory activity incell-free culture liquors was completely inactivated by treatment withtrypsin under the conditions tested. Incorporation of trypsin inhibitorinto the reaction mixture at a concentration of 100 μg/ml prevented thisinactivation. The inhibitory activity was inactivated ca. 50% bytreatment with 100 mg/ml pronase. Higher concentrations of pronase (250μg/ml) or more prolonged treatment (1 h) resulted in completeinactivation of the bacteriocin activity. It appeared to be completelyresistant to inactivation by DNase I, RNase A, lipases, thermolysin, andlysozyme. The proteinaceous nature of the inhibitor indicated by thisexperiment, plus its biological activity, formally qualify it forinclusion in the broad family of bacteriocins. The amino acid sequenceof the subject bacteriocin polypeptide was determined.

EXAMPLE 4 Characterization of Lantibiotic Peptides

[0041] Information on the total number of modified amino acids in alantibiotic can be determined by a combination of a chemicalderivatization and electrospray ionization mass spectroscopy. Edmandegradation of ethane thiol-derivatized mutacin 1140 gave the resultsshown in the following table. This procedure was performed as describedby Mezer et al., (1994) Analyt. Biochem. 223:185-190. TABLE 2 EdmanSequencing of Mutacin 1140 Derivatized with Ethanethiol Cycle PredictedResidue Identified Residue  1 phe phe  2 lys lys  3 ser S-EC^(a)  4 trptrp  5 ser S-EC  6 leu leu  7 cys S-EC  8 thr β-M-S-EC^(a)  9 pro pro 10gly gly 11 cys S-EC 12 ala ala 13 arg arg 14 thr β-M-S-EC 15 gly gly 16ser S-EC 17 phe phe 18 asn asn 19 ser S-EC 20 tyr tyr 21 cys ND^(b) 22cys ND ^(a)Thioethyl cysteine (S-EC) and β-methylthioethyl cysteine(β-M-S-EC) derived from ethanediol derivatization of lanthionine (Lan),3-methyllanthionine (MeLan); 2,3-didehydrolanine (Dha) and2,3-didehydro-2-aminobutyric acid (Dhb) according to the scheme of Myersas presented below:

^(b)Not detected

[0042] These analyses suggested the chemical structure shown in FIGURE1.

EXAMPLE 5 Genetic Analysis

[0043] A genetic analysis of a strain producing the lantibiotic wasperformed. The analysis utilized a plasmid pTV1-OK which is a repA (ts)derivative of the Lactococcus lactis cryptic plasmid pWV01 fortemperature-dependent replication in both Streptococcus mutans andEscherichia coli. The plasmid possesses the transposon Tn917 whichconfers erythromycin resistance in streptococci. Transposon mutagenesiswas performed on lantibiotic-producing strain JH1005 harboring pTV1-OK.Erythromycin resistant clones were selected on BHI agar using 15 μg/mlantibiotic and were then stab inoculated into the same medium withoutantibiotic. After incubation overnight in candle jars at 37° C., theplates were overlaid with 3 ml of top agar containing about 10⁶ colonyforming units per ml of BHT-2. Stabbed clones which failed to producegrowth inhibition of the BHT-2 lawn were recovered and purified bystreaking on a medium with erythromycin.

[0044] From these mutants, which now had the transposon in the geneticelements responsible for lantibiotic production, chromosomal DNA wasisolated and DNA flanking the Tn917 insert was cloned into Escherichiacoli strain MC1061. The flanking DNA was sequenced by the University ofFlorida ICBR using Taq Dye Deoxy Terminator and Dye Primer CycleSequencing protocols as published by Applied Biosystems, using anApplied Biosystems Model 373A DNA Sequencer. Homology searches wereconducted on the recovered sequences using the BLAST program. Therecovered sequences, designated lanA and lanB are presented as SEQ:IDNO:1 and SEQ:ID NO:3 below. These sequences were found to have homologyto epiA and epiB. The open reading frames of these DNA sequences producethe proteins presented in SEQ:ID NO:2 and SEQ:ID NO:4 below.

EXAMPLE 6 Formulation and Administration

[0045] The compounds, polypeptides, and polynucleotides of the inventionare useful for various non-therapeutic and therapeutic purposes. It isapparent from the testing that the compounds, polypeptides, andpolynucleotides of the invention are effective for biochemical probes orcontrolling bacterial growth.

[0046] Therapeutic application of the new compounds and compositionscomprising them can be contemplated to be accomplished by any suitabletherapeutic method and technique presently or prospectively known tothose skilled in the art. Further, the compounds of the invention haveuse as starting materials or intermediates for the preparation of otheruseful compounds and compositions

[0047] The dosage administration to a host in the above indications willbe dependent upon the identity of the infection, the type of hostinvolved, its age, weight, health, kind of concurrent treatment, if any,frequency of treatment, and therapeutic ratio.

[0048] The compounds of the subject invention can be formulatedaccording to known methods for preparing pharmaceutically usefulcompositions. Formulations are described in detail in a number ofsources which are well known and readily available to those skilled inthe art. For example, Remington's Pharmaceutical Science by E. W. Martindescribes formulations which can be used in connection with the subjectinvention. In general, the compositions of the subject invention will beformulated such that an effective amount of the bioactive compound(s) iscombined with a suitable carrier in order to facilitate effectiveadministration of the composition.

[0049] It should be understood that the examples and embodimentsdescribed herein are for illustrative purposes only and that variousmodifications or changes in light thereof will be suggested to personsskilled in the art and are to be included within the spirit and purviewof this application and the scope of the appended claims.

1 5 1 1316 DNA Streptococcus mutans CDS (796)..(987) 1 aatctattttgtagagaatt tagagaaatt attaaattac caagatatgt ttgcaataac 60 atttttaaaatttttaaaaa aaattattac ttactttcat gataagtcag tagatatgtc 120 tgaattagaacattatatta atatagttga agaaataaat cctacgattg cttcaattct 180 taaatctaatttgaatcagc ttttataaag ttttagccat taaagccatc ttgataaatt 240 ttatatctttcatattcatt aaatgtggag ataatgaaaa agcaacggtt atgctatcgc 300 tgctttttttgtgattagaa gctatgttat catggagtta tagtaatgaa acatagtgac 360 agttcatcctttcttattat aaaagtggta ataagagaag tggtaaacaa agagttagta 420 aaataatacgtttaaccata atatttcctc ctttaattta ttataagatt caaaaaggta 480 atattcctatatttgcaaat atgggataaa ataattttaa aaaagcagat ttgcaatttt 540 aaaaaaatagaggctaatgg tggtattata ttattgtaaa tatatgttta ctcagtaata 600 gtgatttactattacaacag attttgttgt tatcttagat atttctgcta gcattagtta 660 tctgtagatgtactacttaa taagtatata attataatta tataataact attatcagat 720 taccgttaaaagttttctga tatgcttcta ctgaacaatt tatgttcagt tacacacatg 780 aaaaaggaggatatt atg tca aac aca caa tta tta gaa gtc ctt ggt act 831 Met Ser AsnThr Gln Leu Leu Glu Val Leu Gly Thr 1 5 10 gaa act ttt gat gtt caa gaagat ctc ttt gct ttt gat aca aca gat 879 Glu Thr Phe Asp Val Gln Glu AspLeu Phe Ala Phe Asp Thr Thr Asp 15 20 25 act act att gtg gca agc aac gacgat cca gat act cgt ttc aaa agt 927 Thr Thr Ile Val Ala Ser Asn Asp AspPro Asp Thr Arg Phe Lys Ser 30 35 40 tgg agc ctt tgt acg cct ggt tgt gcaagg aca ggt agt ttc aat agt 975 Trp Ser Leu Cys Thr Pro Gly Cys Ala ArgThr Gly Ser Phe Asn Ser 45 50 55 60 tac tgt tgc tga ttgtataaaagatttagatt gtgccgcatg ttagcggcac 1027 Tyr Cys Cys aatcttttga tattagaggtattaatatgt taaatacaca attattagaa gtccttggta 1087 ctaaaacttt tgatgttcaagaagatttat ttgagtttaa tataacagat actattgtac 1147 tgcaggctag tgatagtccagatactcata gtaggggtcc cgagcgctta gtgggaattt 1207 gtatcgataa ggggtacaaattcccactaa accaatgttt caaggcctat ttatttttta 1267 tattcaattc tcttaagtgtttaggaatag ataacaagtc aaatttata 1316 2 63 PRT Streptococcus mutans 2 MetSer Asn Thr Gln Leu Leu Glu Val Leu Gly Thr Glu Thr Phe Asp 1 5 10 15Val Gln Glu Asp Leu Phe Ala Phe Asp Thr Thr Asp Thr Thr Ile Val 20 25 30Ala Ser Asn Asp Asp Pro Asp Thr Arg Phe Lys Ser Trp Ser Leu Cys 35 40 45Thr Pro Gly Cys Ala Arg Thr Gly Ser Phe Asn Ser Tyr Cys Cys 50 55 60 31323 DNA Streptococcus mutans CDS (228)..(782) 3 tagtaaagtg ggtagtttcaatatctgccc tcctcgaaag atctccgtca gtttcaatag 60 ttactgttgt taactataaattatacttaa attgatagga aacttggtcg tgacattatc 120 atatgttgat attggaagagaatcaaattt ataaagacaa ttaaatctaa atttgatgaa 180 tatttagatg aattattactaggttgacag tcatgttagg agaagag atg aac gat 236 Met Asn Asp 1 ttt caa tttcaa gat tat ttt atg tac aga aaa cca tta ggc aac ttt 284 Phe Gln Phe GlnAsp Tyr Phe Met Tyr Arg Lys Pro Leu Gly Asn Phe 5 10 15 tct aat ttt tttagt ata act gat acg atg gat ccc att gag tta cta 332 Ser Asn Phe Phe SerIle Thr Asp Thr Met Asp Pro Ile Glu Leu Leu 20 25 30 35 cat agt gat ccgata ttt gct gaa gga gta tat ttg gcc tct tca tct 380 His Ser Asp Pro IlePhe Ala Glu Gly Val Tyr Leu Ala Ser Ser Ser 40 45 50 ctt aga gca gcc ataaat aaa ctt aag aat cat act gcg agt act aag 428 Leu Arg Ala Ala Ile AsnLys Leu Lys Asn His Thr Ala Ser Thr Lys 55 60 65 gat aaa aag aat gca agagag act att ttt caa tac tat gcc cgt tat 476 Asp Lys Lys Asn Ala Arg GluThr Ile Phe Gln Tyr Tyr Ala Arg Tyr 70 75 80 aac acg aga tca act ccg tttggc ttg ttt tcg tcc atc gga gta ggt 524 Asn Thr Arg Ser Thr Pro Phe GlyLeu Phe Ser Ser Ile Gly Val Gly 85 90 95 gct ttt tcg gct tac ctt aaa aaagaa aag tct cgt tat gaa aaa tct 572 Ala Phe Ser Ala Tyr Leu Lys Lys GluLys Ser Arg Tyr Glu Lys Ser 100 105 110 115 att aat att gat ctt ttt tgggct tat aaa gta gca gat aaa cta gaa 620 Ile Asn Ile Asp Leu Phe Trp AlaTyr Lys Val Ala Asp Lys Leu Glu 120 125 130 agt atg cct gaa att tta aatact tta aaa gta gtt gct aat aat gct 668 Ser Met Pro Glu Ile Leu Asn ThrLeu Lys Val Val Ala Asn Asn Ala 135 140 145 ttg caa aag tca gat aat ttttgg ctt ttg gat acg cga agt cat ttt 716 Leu Gln Lys Ser Asp Asn Phe TrpLeu Leu Asp Thr Arg Ser His Phe 150 155 160 ggt ctt atg aat tct ttt catttt atc ttg tac gac ttc tat tct ttc 764 Gly Leu Met Asn Ser Phe His PheIle Leu Tyr Asp Phe Tyr Ser Phe 165 170 175 ctt caa gat aga cca taagaattgatat atcagctgga ttcacaccag 812 Leu Gln Asp Arg Pro 180 aaatacggctagcttgacca atagtttctg ggttaatttt cttaaatttc tgacgtgctt 872 cggtcgcaatagaatcaatg gcatcccaat cgatattctt aggaattcga gctcggtacc 932 cggggatcctctagagtcga cctgcaggca tgcaagcttg gcactggccg tcgttttaca 992 acgtcgtgactgggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc 1052 tttcgccagctggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg 1112 cagcctgaatggcgaatggc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat 1172 ttcacaccgcatatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca 1232 gccccgacacccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc 1292 cgcttacagacaagctgtga ccgtctccgg g 1323 4 184 PRT Streptococcus mutans 4 Met AsnAsp Phe Gln Phe Gln Asp Tyr Phe Met Tyr Arg Lys Pro Leu 1 5 10 15 GlyAsn Phe Ser Asn Phe Phe Ser Ile Thr Asp Thr Met Asp Pro Ile 20 25 30 GluLeu Leu His Ser Asp Pro Ile Phe Ala Glu Gly Val Tyr Leu Ala 35 40 45 SerSer Ser Leu Arg Ala Ala Ile Asn Lys Leu Lys Asn His Thr Ala 50 55 60 SerThr Lys Asp Lys Lys Asn Ala Arg Glu Thr Ile Phe Gln Tyr Tyr 65 70 75 80Ala Arg Tyr Asn Thr Arg Ser Thr Pro Phe Gly Leu Phe Ser Ser Ile 85 90 95Gly Val Gly Ala Phe Ser Ala Tyr Leu Lys Lys Glu Lys Ser Arg Tyr 100 105110 Glu Lys Ser Ile Asn Ile Asp Leu Phe Trp Ala Tyr Lys Val Ala Asp 115120 125 Lys Leu Glu Ser Met Pro Glu Ile Leu Asn Thr Leu Lys Val Val Ala130 135 140 Asn Asn Ala Leu Gln Lys Ser Asp Asn Phe Trp Leu Leu Asp ThrArg 145 150 155 160 Ser His Phe Gly Leu Met Asn Ser Phe His Phe Ile LeuTyr Asp Phe 165 170 175 Tyr Ser Phe Leu Gln Asp Arg Pro 180 5 22 PRTStreptococcus mutans MISC_FEATURE (3)..(3) 2,3-didehydroalanine 5 PheLys Xaa Trp Xaa Leu Xaa Xaa Pro Gly Xaa Ala Arg Xaa Gly Xaa 1 5 10 15Phe Asn Xaa Tyr Xaa Xaa 20

I claim:
 1. An isolated nucleic acid comprising the nucleotide sequenceof SEQ ID NO:3.
 2. An isolated nucleic acid comprising a sequence thatencodes a polypeptide with the amino acid sequence of SEQ ID NO:4.
 3. Abacterial host transformed with the nucleic acid of claim
 2. 4. Thebacterial host of claim 3, wherein the bacterial host is a Streptococcusspecies.
 5. A purified polypeptide comprising the amino acid sequenceshown in SEQ ID NO:4.